Physical activity is vital in the maintenance of glucose homeostasis in people with type 2 diabetes. The molecular signaling mechanisms by which exercise regulates glucose metabolism in skeletal muscle are not fully understood. AMP-activated protein kinase (AMPK) is considered a master metabolic regulator in muscle and other tissues, and is activated by exercise in skeletal muscle. The tumor suppressor protein LKB1 has recently been identified as an upstream kinase for AMPK and 12 other AMPK-related proteins. To elucidate the expanding role of AMPK and LKB1 in muscle and their potential use in therapeutic applications, we have studied muscle metabolism using multiple mouse models of altered AMPK and LKB1 activity. AMPK is now known not the only mediator of contraction-stimulated glucose uptake in skeletal muscle. Instead, our findings show that LKB1, acting through AMPK and AMPK-related kinases, plays a central role in this process. Increased post-exercise insulin sensitivity is an important component of exercise-related metabolic adaptations in muscle; our data suggest that both LKB1 and AMPK mediate this increase. We also show here that LKB1 regulates the expression of Tribbles 3 (TRB3) in skeletal muscle. Mice overexpressing TRB3 in skeletal muscle have dramatically increased exercise capacity, and skeletal muscle TRB3 is increased with exercise training. Hence, while the major focus of this project has been the study of AMPK in the regulation of muscle metabolism, it has expanded to include LKB1 and its other downstream proteins. Our hypothesis is that LKB1 is a central regulator of acute and chronic metabolic responses to exercise in skeletal muscle in conjunction with AMPK and other downstream proteins. Specific Aim 1 will determine the role of AMPK-related kinases in contraction-stimulated skeletal muscle glucose uptake. We will test the hypothesis that AMPK-related kinases SNARK ARK5, SIK1 and MARK4 regulate glucose uptake by using novel techniques including mutagenesis and in vivo gene transfer into adult mouse skeletal muscle. Specific Aim 2 will determine the role of LKB1 and AMPK in post-exercise increases in insulin sensitivity for glucose uptake and insulin signaling. We will also determine how AMPK regulates the temporal and spatial dynamics of GLUT4 translocation in the before and after exercise using a novel imaging method that has not previously been used in this context. Specific Aim 3 will determine the role of TRB3 in skeletal muscle exercise metabolism. One of the most striking features of the muscle-specific LKB1 knockout mice is that they have a dramatic reduction in TRB3 expression in skeletal muscle. We have generated exciting data showing that transgenic mice overexpressing TRB3 in skeletal muscle have dramatically increased exercise capacity and altered of fiber type composition. These findings implicate TRB3 as an essential mediator of metabolic adaptations in skeletal muscle. Collectively, our studies will enhance the understanding of contraction-induced alterations in skeletal muscle glucose metabolism. PUBLIC HEALTH RELEVANCE: Exercise performed on a regular basis can delay the onset as well as reduce the insulin resistance associated with type 2 diabetes. Because the rate of diabetes has the potential to reach epidemic proportions in the near future, our proposed research is dedicated to determining the molecular mechanisms involved in the positive effects of exercise in skeletal muscle. We have identified several key proteins (e.g. LKB1, SNARK, TRB3) that are likely to be important in mediating the beneficial effects of exercise and their elucidation could lead to the development of novel therapeutic options for individuals with type 2 diabetes.